RESUMEN
Environmental fluctuations are a common challenge for single-celled organisms; enteric bacteria such as Escherichia coli experience dramatic changes in nutrient availability, pH, and temperature during their journey into and out of the host. While the effects of altered nutrient availability on gene expression and protein synthesis are well known, their impacts on cytoplasmic dynamics and cell morphology have been largely overlooked. Here, we discover that depletion of utilizable nutrients results in shrinkage of E. coli's inner membrane from the cell wall. Shrinkage was accompanied by an â¼17% reduction in cytoplasmic volume and a concurrent increase in periplasmic volume. Inner membrane retraction after sudden starvation occurred almost exclusively at the new cell pole. This phenomenon was distinct from turgor-mediated plasmolysis and independent of new transcription, translation, or canonical starvation-sensing pathways. Cytoplasmic dry-mass density increased during shrinkage, suggesting that it is driven primarily by loss of water. Shrinkage was reversible: upon a shift to nutrient-rich medium, expansion started almost immediately at a rate dependent on carbon source quality. A robust entry into and recovery from shrinkage required the Tol-Pal system, highlighting the importance of envelope coupling during shrinkage and recovery. Klebsiella pneumoniae also exhibited shrinkage when shifted to carbon-free conditions, suggesting a conserved phenomenon. These findings demonstrate that even when Gram-negative bacterial growth is arrested, cell morphology and physiology are still dynamic.
Asunto(s)
Citoplasma/fisiología , Escherichia coli/fisiología , Carbono/deficiencia , Carbono/farmacología , Citoplasma/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/metabolismo , Canales Iónicos/metabolismo , Mecanotransducción Celular/efectos de los fármacos , Nitrógeno/análisis , Fósforo/análisisRESUMEN
Bacteria frequently encounter nutrient fluctuations in natural environments, yet we understand little about their ability to maintain physiological memory of previous food sources. Starvation is a particularly acute case, in which cells must balance adaptation to stresses with limited nutrient supply. Here, we show that Escherichia coli cells immediately accelerate and decelerate in growth upon transitions from spent to fresh media and vice versa, respectively, and memory of rapid growth can be maintained for many hours under constant flow of spent medium. However, after transient exposure of stationary-phase cells to fresh medium, subsequent aerobic incubation in increasingly spent medium led to lysis and limited growth when rejuvenated in fresh medium. Growth defects were avoided by incubation in anaerobic spent medium or water, suggesting that defects were caused by respiration during the process of nutrient depletion in spent medium. These findings highlight the importance of respiration for stationary phase survival and underscore the broad range of starvation outcomes depending on environmental history.
RESUMEN
Bacterial survival is often challenged by nutrient-depleted conditions. Here, we show that Escherichia coli regrowth from prolonged stationary phase is heterogeneous. Some cells rejuvenated immediately, even after extended starvation, but others only restarted growth after a delay or not at all. The proportion of nongrowing cells increased with time spent in stationary phase, rather than time-dependent medium changes. Delayed regrowth was correlated with the dissolution of polar phase-bright foci likely representing damaged protein aggregates, and a deep learning algorithm distinguished cellular fates based on single images. Delayed regrowth initiated after upregulation of chaperones and DNA-repair enzymes, and deletion of a chaperone compromised stationary-phase morphology and increased the nongrowing cell proportion. Mathematical modeling of damage accumulation and division-mediated partitioning quantitatively predicted all rejuvenation statistics. Cells regrew immediately after starving in the absence of respiration. These findings reinforce the importance of intracellular damage control when nutrients are sparse, and repair when nutrients are plentiful.
RESUMEN
CRISPR interference (CRISPRi) has facilitated the study of essential genes in diverse organisms using both high-throughput and targeted approaches. Despite the promise of this technique, no comprehensive arrayed CRISPRi library targeting essential genes exists for the model bacterium Escherichia coli, or for any Gram-negative species. Here, we built and characterized such a library. Each of the â¼500 strains in our E. coli library contains an inducible, chromosomally integrated single guide RNA (sgRNA) targeting an essential (or selected nonessential) gene and can be mated with a pseudo-Hfr donor strain carrying a dcas9 cassette to create a CRISPRi knockdown strain. Using this system, we built an arrayed library of CRISPRi strains and performed population and single-cell growth and morphology measurements as well as targeted follow-up experiments. These studies found that inhibiting translation causes an extended lag phase, identified new modulators of cell morphology, and revealed that the morphogene mreB is subject to transcriptional feedback regulation, which is critical for the maintenance of morphology. Our findings highlight canonical and noncanonical roles for essential genes in numerous aspects of cellular homeostasis. IMPORTANCE Essential genes make up only â¼5 to 10% of the genetic complement in most organisms but occupy much of their protein synthesis and account for almost all antibiotic targets. Despite the importance of essential genes, their intractability has, until recently, hampered efforts to study them. CRISPRi has facilitated the study of essential genes by allowing inducible and titratable depletion. However, all large-scale CRISPRi studies in Gram-negative bacteria thus far have used plasmids to express CRISPRi components and have been constructed in pools, limiting their utility for targeted assays and complicating the determination of antibiotic effects. Here, we use a modular method to construct an arrayed library of chromosomally integrated CRISPRi strains targeting the essential genes of the model bacterium Escherichia coli. This library enables targeted studies of essential gene depletions and high-throughput determination of antibiotic targets and facilitates studies targeting the outer membrane, an essential component that serves as the major barrier to antibiotics.
Asunto(s)
Sistemas CRISPR-Cas , Escherichia coli/genética , Técnicas de Silenciamiento del Gen/métodos , Biblioteca de Genes , Genes Esenciales/genética , Transcripción Genética , Proteínas Bacterianas/metabolismo , Ensayos Analíticos de Alto RendimientoRESUMEN
Bacteria must maintain a cytosolic osmolarity higher than that of their environment in order to take up water. High-osmolarity environments therefore present formidable stress to bacteria. To explore the evolutionary mechanisms by which bacteria adapt to high-osmolarity environments, we selected Escherichia coli in media with a variety of osmolytes and concentrations for 250 generations. Adaptation was osmolyte dependent, with sorbitol stress generally resulting in increased fitness under conditions with higher osmolarity, while selection in high concentrations of proline resulted in increased fitness specifically on proline. Consistent with these phenotypes, sequencing of the evolved populations showed that passaging in proline resulted in specific mutations in an associated metabolic pathway that increased the ability to utilize proline for growth, while evolution in sorbitol resulted in mutations in many different genes that generally resulted in improved growth under high-osmolarity conditions at the expense of growth at low osmolarity. High osmolarity decreased the growth rate but increased the mean cell volume compared with growth on proline as the sole carbon source, demonstrating that osmolarity-induced changes in growth rate and cell size follow an orthogonal relationship from the classical Growth Law relating cell size and nutrient quality. Isolates from a sorbitol-evolved population that captured the likely temporal sequence of mutations revealed by metagenomic sequencing demonstrated a trade-off between growth at high osmolarity and growth at low osmolarity. Our report highlights the utility of experimental evolution for dissecting complex cellular networks and environmental interactions, particularly in the case of behaviors that can involve both specific and general metabolic stressors.IMPORTANCE For bacteria, maintaining higher internal solute concentrations than those present in the environment allows cells to take up water. As a result, survival is challenging in high-osmolarity environments. To investigate how bacteria adapt to high-osmolarity environments, we maintained Escherichia coli in a variety of high-osmolarity solutions for hundreds of generations. We found that the evolved populations adopted different strategies to improve their growth rates depending on the osmotic passaging condition, either generally adapting to high-osmolarity conditions or better metabolizing the osmolyte as a carbon source. Single-cell imaging demonstrated that enhanced fitness was coupled to faster growth, and metagenomic sequencing revealed mutations that reflected growth trade-offs across osmolarities. Our study demonstrated the utility of long-term evolution experiments for probing adaptation occurring during environmental stress.
Asunto(s)
Adaptación Fisiológica/genética , Escherichia coli/genética , Evolución Molecular , Mutación , Estrés Fisiológico/genética , Recuento de Colonia Microbiana , Medios de Cultivo/química , Ambiente , Escherichia coli/crecimiento & desarrollo , Concentración Osmolar , FenotipoRESUMEN
Bacterial growth under nutrient-rich and starvation conditions is intrinsically tied to the environmental history and physiological state of the population. While high-throughput technologies have enabled rapid analyses of mutant libraries, technical and biological challenges complicate data collection and interpretation. Here, we present a framework for the execution and analysis of growth measurements with improved accuracy over that of standard approaches. Using this framework, we demonstrate key biological insights that emerge from consideration of culturing conditions and history. We determined that quantification of the background absorbance in each well of a multiwell plate is critical for accurate measurements of maximal growth rate. Using mathematical modeling, we demonstrated that maximal growth rate is dependent on initial cell density, which distorts comparisons across strains with variable lag properties. We established a multiple-passage protocol that alleviates the substantial effects of glycerol on growth in carbon-poor media, and we tracked growth rate-mediated fitness increases observed during a long-term evolution of Escherichia coli in low glucose concentrations. Finally, we showed that growth of Bacillus subtilis in the presence of glycerol induces a long lag in the next passage due to inhibition of a large fraction of the population. Transposon mutagenesis linked this phenotype to the incorporation of glycerol into lipoteichoic acids, revealing a new role for these envelope components in resuming growth after starvation. Together, our investigations underscore the complex physiology of bacteria during bulk passaging and the importance of robust strategies to understand and quantify growth.IMPORTANCE How starved bacteria adapt and multiply under replete nutrient conditions is intimately linked to their history of previous growth, their physiological state, and the surrounding environment. While automated equipment has enabled high-throughput growth measurements, data interpretation and knowledge gaps regarding the determinants of growth kinetics complicate comparisons between strains. Here, we present a framework for growth measurements that improves accuracy and attenuates the effects of growth history. We determined that background absorbance quantification and multiple passaging cycles allow for accurate growth rate measurements even in carbon-poor media, which we used to reveal growth-rate increases during long-term laboratory evolution of Escherichia coli Using mathematical modeling, we showed that maximum growth rate depends on initial cell density. Finally, we demonstrated that growth of Bacillus subtilis with glycerol inhibits the future growth of most of the population, due to lipoteichoic acid synthesis. These studies highlight the challenges of accurate quantification of bacterial growth behaviors.
Asunto(s)
Adaptación Fisiológica , Ambiente , Escherichia coli/crecimiento & desarrollo , Bacillus subtilis/crecimiento & desarrollo , Medios de Cultivo/farmacología , Glicerol/farmacología , Modelos Teóricos , FenotipoRESUMEN
The determination of cell size is a fundamental challenge for all living organisms. In a given growth condition, cell size for a particular bacterial species typically falls within a narrow distribution. Nonetheless, size can vary enormously across species, and the size of a single bacterium can even vary substantially across growth conditions. Recent phenomenological studies have revived classic interest in how cells maintain their size and how they adjust their size with changes in growth rate. However, the mechanisms by which cells establish a particular size are relatively enigmatic. Here, we review existing knowledge on how size in rod-shaped bacteria is shaped by nutrient, mechanical, and genetic factors. We also examine obstacles to accurate size measurement and recent technologies that help to overcome these hurdles. Finally, we discuss the relevance of cell size to bacterial physiology.
Asunto(s)
Bacterias/citología , Fenómenos Fisiológicos Bacterianos , Bacterias/genética , Bacterias/crecimiento & desarrolloRESUMEN
Bacteria must maintain mechanical integrity to withstand the large osmotic pressure differential across the cell membrane and wall. Although maintaining mechanical integrity is critical for proper cellular function, a fact exploited by prominent cell-wall-targeting antibiotics, the proteins that contribute to cellular mechanics remain unidentified. Here, we describe a high-throughput optical method for quantifying cell stiffness and apply this technique to a genome-wide collection of â¼4,000 Escherichia coli mutants. We identify genes with roles in diverse functional processes spanning cell-wall synthesis, energy production, and DNA replication and repair that significantly change cell stiffness when deleted. We observe that proteins with biochemically redundant roles in cell-wall synthesis exhibit different stiffness defects when deleted. Correlating our data with chemical screens reveals that reducing membrane potential generally increases cell stiffness. In total, our work demonstrates that bacterial cell stiffness is a property of both the cell wall and broader cell physiology and lays the groundwork for future systematic studies of mechanoregulation.